Characterization and control of Rhizoctonia solani affecting lucky bamboo (Dracaena sanderiana hort. ex. Mast.) using some bioagents

In a survey conducted during the period of March–May 2019 in nurseries, warehouses, and shops at three governorates (Alexandria, El-Behera, and Giza governorates, Egypt), symptoms of root rot, basal stem rot, and wilt disease complex were observed in the lucky bamboo (Dracaena sanderiana hort. ex. Mast.). The highest disease infection percentage was found in lucky bamboo collected from Alexandria City (47.67%), while the highest disease severity was in lucky bamboo collected from El-Behera Governorate (35.19%). Rhizoctonia solani, Fusarium oxysporum, F. solani, Aspergillus niger, and Alternaria alternate were isolated and identified in the infected lucky bamboo samples. R. solani isolates were the most dominant among the recovered fungal species with a percentage of 80.89% of the total isolates (246). Pathogenicity tests showed that R. solani was the most pathogen with 100% disease infection and 76.67% disease severity. Molecular identification characterized R. solani isolate as R. solani AUMC 15120, MZ723906. Meanwhile, four biological control agents (bioagents) were isolated from the healthy lucky bamboo samples and identified based on cultural, morphological, microscopic characteristics, and the molecular phylogenetic analysis as Clonostachys rosea AUMC 15121, OL461708; Bacillus circulans TAG1, MW441316; B. siamensis TAP1, MW441318 and Ochrobactrum anthropi TAM1, MW441317. The four bioagents showed potential inhibition of R. solani in vitro as well as in vivo on lucky bamboo plants in vase treatments compared to the untreated inoculated control as well as certain fungicides and biocides used (Moncut, Rizolex-T, Topsin-M, Bio-Zeid, and Bio-Arc). The bioagent O. anthropi showed the highest inhibition growth (85.11%) of the in vitro R. solani colony, which was not significantly different from the biocide Bio-Arc (83.78%). However, C. rosea, B. siamensis and B. circulans showed inhibition values of 65.33, 64.44, and 60.44%, respectively. On the other hand, the biocide Bio-Zeid showed less inhibitory effect (43.11%), while the lowest growth inhibition was recorded by Rizolex-T (34.22%) and Topsin-M (28.67%). Furthermore, the in vivo experiment supported the in vitro results for the most effective treatments, where all the treatments significantly decreased the percentage of infection and disease severity compared to the inoculated untreated control. Additionally, the bioagent O. anthropi showed the highest effect, i.e., the lowest disease incidence and disease severity being 13.33% and 10%, compared to 100% and 75%, respectively, in the untreated inoculated control. This was not significantly different from the fungicide Moncut (13.33% and 21%) and from the bioagent C. rosea (20% and 15%) treatments for both parameters, respectively. In conclusion, the bioagents O. anthropi MW441317 at 1 × 108 CFU/ml as well as C. rosea AUMC15121 at 1 × 107/ml proved to be efficient to control R. solani causing root rot, and basal stem rot on lucky bamboo, compared to fungicide Moncut and can be used for disease management without the negative impact of the chemical control. Furthermore, this is the first report of the isolation and identification of Rhizoctonia solani, a pathogenic fungus, and four biocontrol agents (Bacillus circulans, B. siamensis, Ochrobactrum anthropi and Clonostachys rosea) associated with the healthy lucky bamboo plants.

Survey for root rot, basal stem rot, and wilt disease complex on lucky bamboo. During the period of March-May 2019, 226 samples of naturally infected lucky bamboo showing characteristic symptoms of severe root rot, basal stem rot, and wilt disease complex were randomly collected from 630 lucky bamboo plants surveyed at various locations at retail stores, shops, and nurseries in Alexandria, El-Behera, and Giza governorates, Egypt. In all the mentioned sites, the percentages of disease infection were recorded and calculated 30,31 using the ratings shown in Table 1.
The percentages of disease infection and disease severity were calculated by the following equations: samples showing symptoms of root rot, stem basal rot and wilt disease complex were collected in the survey, labeled separately in paper bags, and transferred to the laboratory to isolate the associated fungal species on the day following collection. Each sample was thoroughly washed in running tap water and cut into small pieces (4 mm), with half of the tissue being healthy and half being diseased tissue. The surface of the pieces was sterilized by soaking in sodium hypochlorite (5%) for 3 min after being rinsed with sterile distilled water (DW). The pieces were washed thrice with sterile DW, then dried between two layers of sterilized filter papers, and subsequently placed in Petri dishes with potato dextrose agar (PDA) medium supplemented with 250 µL/mL of streptomycin (Egypt Masters Co. (EMC), Dakahlia, Egypt). The plates were then incubated in darkness at 26 ± 1 °C for 7 days 32 , and the developed fungal colonies were purified by a single spore or hyphal tip technique. The developed fungal species were identified based on morphological and microscopic characteristics [33][34][35] . The frequency of each fungal isolate was calculated according to the following equation.

Pathogenicity tests and molecular identification of the isolated fungal species. Pathogenicity
tests. Ostensibly healthy-looking lucky bamboo plants, with uniform stem lengths averaging 70 cm, were purchased from a famous private commercial nursery in Cairo. In the laboratory, plants were well-washed several times with running tap water, surface disinfected in 1% sodium hypochlorite for 2 min, then washed several times with running tap water and rinsed with sterilized DW. To ensure that the plants are healthy and free of any pathogens, they were sown in glassware containing 300 ml of sterile DW for 60 days under the laboratory conditions (12 h photoperiod at 26 ± 2 °C with an average relative humidity of 65-70%) before conducting any experiments on them. Furthermore, to reduce bacteria entry from the surrounding air and prevent water evaporation, all glassware is wrapped with a sterilized cotton stopper around the bamboo stem. Before sown, each glassware was cleaned and sterilized in a hot air oven for 2 h at 180 °C and then left to cool 3,36 .
Thirty healthy bamboo plants were placed in 1-L sterile glassware (1 plant/glassware) by dipping 5 cm of basal stems with 300 ml of sterile DW. Fifteen plants were inoculated separately by adding directly an excerpt of Rhizoctonia solani mycelial agar plug (0.5 cm diameter) cut from a 7-day-old culture disc at 26 ± 2 °C of the active margins of the fungal culture recovered in the survey. The culture was inserted into a cut in the basal stem segment with a sterile Cork borer. A similar plug of sterile PDA served as the negative control of the remaining 15 bamboo plants. The inoculated areas were then covered with Parafilm strips and to provide wet conditions, the plants were covered with polyethylene plastic bags for 24 h. All bamboo plants were kept for 4 weeks under laboratory conditions. At the end of the test, the percentages of infection and severity of disease were calculated as described above 30,37 .
To ensure that the pathogen was associated with the symptoms, it was re-isolated from the symptoms in artificially infected plant tissues. Subsequently, the developed fungal cultures were purified as described above, then they were identified based on morphological and microscopic characteristics and molecular identification.
Molecular identification of the recovered Rhizoctonia solani isolates. The most dominant fungal species, i.e., R. solani, was further identified and molecularly characterized by polymerase chain reaction (PCR) amplification and 18S sequencing. The cultures were sent to the "Molecular Biology Research Unit, Assiut University for DNA extraction using a Patho Gene-spin DNA/RNA extraction Kit provided by Intron Biotechnology Company, Korea. Samples of fungal DNA were then sent to SolGent Company, Daejeon, South Korea for PCR and 18S sequencing. PCR was performed using ITS1 (forward) and ITS4 (reverse) primers, which were incorporated into the reaction mixture 38 . Primers have areas with universal primer pairs including ITS1 (5'-TCC GTA GGT GAA CCT GCG G-3'), and ITS4 (5'-TCC TCC GCT TAT TGA TAT GC-3'). The purified PCR products (amplicons) were sequenced using the same primers, but with ddNTPs added to the reaction mixture 39 The diseased spots at the base of the stem account for less than 25% of the entire stem circumference and root  discolorations   2  Moderate infections of more than 25%  The diseased spots at the base of the stem account for more than 25% of the entire stem circumference and root  discoloration and/or one leaf yellowed   3 Severe infections for more than 50% The diseased spots at the base of the stem account for more than 50% of the entire stem circumference, and root discoloration and/or vascular discoloration, add 2 ± 1 leaf yellowed and/or 1 ± 1 leaf wilted 4 Very severe infections covering more than 75% The diseased spots at the base of the stem account for more than 75% of the entire stem circumference and root discolorations and/or more than one leaf wilted or completely dead plants Control studies. Isolation of the associated biocontrol agents of the healthy lucky bamboo. Isolation of the antagonistic micro-organisms on healthy lucky bamboo plants was conducted after the incubation period of bamboo plants, which exceeded 60 days under the laboratory conditions, to ensure that they are healthy plants free of any apparent infestations 3 . Plant parts of 2 cm long were cut from the basal stem with roots of 20 bamboo plants, then 1 g of each sample was taken and processed as mentioned above for the isolation and microscopic and molecular identification of the fungi. The isolation of the associated bacteria was conducted according to Abdel-Rahman 40 , with minor modifications. The crushed plant parts were sterilized with sodium hypochlorite solution (10%) for 2 min, immersed in sterilized DW for 2 min, and washed thoroughly several times with sterilized DW. Afterward, 1 g from each sample tissue (parts of each basal stem and root) was mixed with 9.9 ml of sterile saline (sterile physiological water from NaCl, 9 g/l) individually and squashed into the sterilized mortar and pestle to homogenize. Then, the solution was diluted serially in the sterile saline individually for each sample up to 10 6 CFU/ml 41 . A loopful (1 ml) of the resulting suspension of each dilution was spread on nutrient agar (9 ml of NA) plates and incubated for 24 h at 30 ± 1 °C. In order to obtain new separate colonies, single colonies were selected and purified on fresh NA plates on the base of variance in morphology, e.g., color, size, and shape. After 24-48 h at the same temperature, pure bacterial colonies appeared, then identified according to their morphological and biochemical characteristics 42,43 , and performed using standard methods by Agricultural Laboratories Company (Agro Lab, Sadat City, Egypt).
Molecular identification of the associated biocontrol agents of the healthy lucky bamboo. DNA was extracted from the isolation of pure cultures of fungi and bacteria ABT DNA mini extraction Kit (Applied Biotechnology Co. Ltd, Egypt) for molecular characterization of the Internal Transcript Spacer (ITS) region using Polymerase Chain Reaction (PCR) amplification, "2X Red master Mix (Applied Biotechnology Co., Egypt), and Oligonucleotide (Alpha DNA Co, Canada)" 44 . The ITS DNA region of these isolates was amplified via PCR using universal primers. The following is the optimized thermal profile for PCR: Initial denaturation (95 °C for 3 min), Denaturation (95 °C for 30 s., Annealing (50 °C for 30 s.), Extension (72 °C for 90 s.), and Final extension (72 °C for 5 min.), Repeating for 35 cycles.
To confirm the targeted PCR amplification, five μl of the PCR product was electrophoresed along with 100 bp DNA molecular weight 1% agarose gel containing ethidium bromide (0.5 μg/ml) at constant 80 V for 30 min in 1X TAE buffer. The amplified product was visualized as a single compact band of expected size under UV light and documented by the Samsung Note 4 smartphone.
Sequencing of the PCR product for the amplified PCR products was submitted to Solgent Co Ltd (South Korea) for gel purification and sequencing. The resulted sequences were trimmed and assembled in Geneious software (Biomatters). Consequently, the trimmed sequences were identified by search in the basic local alignment search tool (BLAST) in GenBank.
Furthermore, phylogenetic analysis by nucleotide sequences was downloaded from GenBank and aligned with the identified sequences, using MAFFT alignment 45 . Phylogenetic trees were constructed using the neighborjoining method, employing the Tamura-Nei Model 46 . The trees were assessed using 1000 bootstrap replicates.
Evaluation of the isolated bioagents against Rhizoctonia solani in vitro. The in vitro inhibition effects of the recovered bioagent isolates, i.e., the fungal isolate Clonostachys rosea, and the three recovered bacterial isolates Bacillus circulans, B. siamensis, and Ochrobactrum anthropi, were tested against the highly aggressive isolate of R. solani AUMC15120, which recovered in the conducted survey. This was done in comparison with the untreated inoculated control as a negative control as well as three fungicides and two biocides as positive controls ( Table 2).
The tested fungal and bacterial bioagents as well as the tested fungal and bacterial biocides were grown under laboratory conditions. For biocides, a disc of filter paper 5 mm in diameter, impregnated with Bio-Zeid (Trichoderma album 25 × 10 6 spores/g) suspension, was inoculated at the recommended rate into the middle of a Petri dish, then incubated at 26 ± 2 °C for 7-days, while; bacterial isolates and/or Bio-Arc were grown individually in 250 ml flasks that each contained 50 ml of the NA medium. Then after the incubation for 72 h, they were used for the streaked method.
Antagonistic potential of the isolated bioagents against Rhizoctonia solani. Isolated bioagents were screened for their antagonistic potential against R. solani by double culture assay using solid PDA plates 50 . Each PDA plate was divided into two equal halves, and at a distance of 1 cm from the edge of every plate from opposite sides, plates were inoculated on one side with a mycelial disc (5 mm diameter) taken from the margins of the active growing R. solani of 7-day-old PDA cultures. Furthermore, on the other opposite side of each plate at 1 cm distance from the plate edge, a 5 mm of the tested fungal bioagent (C. rosea) or fungal biocide plug (taken from advanced margins of 7-day-old PDA cultures) was placed.
Likewise, a filter paper disc of 5 mm diameter, impregnated with each fungicide separately was placed 51 . Additionally, the bacterial bioagent isolates and/or Bio-Arc were treated by a streaking method 3 . Untreated check (control) plates were treated without bioagents and/or fungicides. www.nature.com/scientificreports/ Five replicate plates were used for each treatment and incubated at 26 ± 2 °C until the untreated control mycelium (R. solani) totally colonized the plate. At that time, radial growth in plates for each treatment was measured to determine the percentage of the growth inhibition by calculating the percentage of radial growth reduction in diameter mycelia of R. solani 3,50 as follows 3,44 : In which (GD u ) is the radial growth diameter of pathogen mycelia untreated (R. solani) in the control plate, apart from the bioagents (cm), and (GD T ) is the radial growth diameter of the treated pathogen mycelia (R. solani) toward the bioagents and/or fungicides (cm).  Table 2) that showed maximum inhibitory activity in vitro against the highly aggressive R. solani AUMC15120 isolate. i.e., Clonostachys rosea AUMC15121″, Bacillus circulans MW441316, B. siamensis MW441318, Ochrobactrum anthropi MW441316, Moncut, Bio-Zeid and Bio-Arc were tested in vivo on lucky bamboo in the vase under laboratory conditions at the recommended dose ( Table 2).

The in vivo evaluation of the efficacy of the isolated bioagents to control
Inocula of the fungal bioagent C. rosea were prepared as follows; flasks of 100 ml of potato dextrose liquid (PDL) were inoculated by adding directly a sporulating mycelial agar plug (0.5 cm diameter) taken from a 7-day-old culture active margins of C. rosea and incubated at 22 ± 2 °C with photoperiods 12 h under white fluorescent lamps until sufficient mycelial growth was obtained. Subsequently, the collected mycelial growth was blended with 100 ml of sterile distilled water for 1 min. The conidia spore's concentration was adjusted using the hemocytometer technique 47 , viz 10 7 conidia spores/ml inoculum concentrations.
The bacterial suspensions of bioagents and biocides were cultured on Luria-Bertani (LB) broth medium at flasks containing 100 ml of medium for 2 days at 30 ± 1 °C and shaken at 200 rpm to harvest bacteria. Then the bacterial concentrations in the solution were adjusted by serial dilution with sterile saline individually 1 × 10 7 CFU/ml 48 , and 1 × 10 8 CFU/ml 49 .
All treatment were conducted one day after inoculation with R. solani (as mentioned above in the pathogenicity testing) to 120 glassware vessels planted with healthy lucky bamboo separately. Sterile distilled water was used instead of the treatments as an untreated control. All treatments and control were kept for 4 weeks under laboratory conditions. At the end of the trial, the percentages of disease infection and disease severity (%) were recorded as mentioned above. Subsequently, the percentage decrease or increase from the untreated control was calculated 52 . In addition, the percentage of treatment efficiency was evaluated using the formulas proposed 53 , respectively: Decrease  www.nature.com/scientificreports/ Statistical analysis. The obtained data of the tested treatments (bioagents, fungicides, and biocides) against the growth of R. solani were statistically analyzed using Statistix program, by using the software analysis of variance with one-way ANOVA test 54 and compared with the untreated control. All trials were carried out with a randomized complete block design (RCBD). Each treatment with five replicates, each replicate contains 3 plates and/or 3 glassware. Each glassware contains one bamboo plant. Comparisons among the means were evaluated using the least significance difference (LSD) at a 5% level of probability.

Results
Survey for root rot, basal stem rot and wilt disease complex on lucky bamboo. During the period March-May 2019, the conducted survey for root rot, basal stem rot and wilt disease complex on lucky bamboo plants, typical symptoms were identified (Fig. 1) in the surveyed retail stores, shops, and nurseries from different locations in Egypt (Alexandria, El-Behera, and Giza governorates). However, the surveyed locations and governorates showed considerable variations in the percentage of infection and disease severity % (Table 3). From the data in Table 3, the mean percentage of disease infection was the highest in plants collected from Alexandria (47.67%) followed by Giza (33.14%) and El-Behera (26.98%) Governorates. However, the disease severity for the three surveyed governorates ranged between 35.19and 31.53%, with no significant differences between the surveyed governorates ( Table 3).  Table 4 show that the five fungal isolates R. solani, F. oxysporum, F. solani, Aspergillus niger, and Alternaria alternata were found to be associated with the surveyed lucky bamboo samples with symptoms of root rot, basal stem rot, and wilt disease complex. However, R. solani isolates were the most frequent among fungal species recovered and constituted 80.89% of the total isolates while the other fungal species showed frequencies lower than 10% (Fig. 2). Table 5 that the tested fungal species recovered in the survey were able to incite root rot and basal stem rot to different degrees. However, data in Table 5 (Table 5). Moreover, Fig. 3 illustrated the symptoms of basal stem rot and root rot when artificial infestation by R. solani during the pathogenicity test and compared it with that of natural infestation of lucky bamboo plants.  Table 4. The overall mean of frequent fungal species recovers isolated from diseased root rot and basal stem rot of lucky bamboo after beingsurveyed from different locations in the three Egyptian governorates.  (Fig. 4), the molecular identification of R. solani isolates was conducted by PCR amplification and 18S sequencing. The tested strain showed 100% identity and 100% coverage with several strains of R. solani accessed from the GenBank (Fig. 5). The fungus was putatively identified as Rhizoctonia solani AUMC 15120 (GenBank accession No. MZ723906). Furthermore, the phylogenetic tree identified showed that R. solani (AUMC 15120) aligned with closely related sequences accessed from the GenBank, i.e., Thanatephorus cucumeris which is the teleomorph (sexual stage) of R. solani (Fig. 5).

Control studies. Isolation and identification of the associated biocontrol agents of the healthy lucky bam-
boo. Four biological control agents were isolated from healthy lucky bamboo plants ( Table 6). Three of them were bacterial strains (Bacillus circulans, B. siamensis, and Ochrobactrum anthropi), and one fungal strain (Clonostachys rosea). The isolated fungus was morphologically investigated (Fig. 6) and all bioagents were characterized at the molecular level ( Figs. 7 and 8).
The in vitro evaluation of the isolated bioagents against Rhizoctonia solani. The isolated bioagents from healthy lucky bamboo were evaluated for their antagonistic potential against R. solani recovered from lucky bamboo, in vitro and in vivo and compared with some other fungicides and biocides.
It is evident in Fig. 9 that all tested bioagents and the tested fungicides and biocides affected the tested R. solani isolate to different degrees. However, data in Table 7 showed that the highest R. solani colony growth inhibition    (Table 7).

The in vivo evaluation of the efficacy of the isolated bioagents to control Rhizoctonia solani on lucky bamboo in
vase under laboratory conditions. The most effective treatments revealed in vitro were further tested in vivo in vases under laboratory conditions. Data in Table 8 showed the disease incidence and disease severity caused by R. solani. A decrease or increase in infection with and treatment effectiveness against R. solani on bamboo artificially inoculated under laboratory conditions for 4 weeks after inoculation and treatments are shown in Fig. 10. All the tested treatments (Table 8) significantly decreased the infection percentage and disease severity compared to the inoculated untreated control. However, the best effect was obtained by O. anthropi and the fungicide Moncut, with the lowest disease incidence value of 13.33%, followed by C. rosea (20%) compared to the control (100%). The lowest disease severity (10%) was obtained by O. anthropi followed by Clonostachys rosea (15%) and B. siamensis (15%) compared to the control (75%). However, B. circulans showed 40% and 27% while, Bio-Zeid (Trichoderma album) showed the least effect with values of 66.67% and 43.33%, for both disease parameters, respectively (Table 8).
Furthermore, all the tested treatments significantly decreased the disease incidence and gave positive increasing responses regarding the treatment efficiency in comparison to the untreated control (Fig. 10). O. anthropi afforded the highest disease reduction to other isolates, and it gave the highest efficiency at 86.67%. Bio-Zeid (T. album) yielded the lowest reduction of the pathogen and the lowest mean percentage of efficiency (viz 33.33 and 42.22%, respectively, both disease parameters) compared to the infected untreated control.   The present study confirmed these reports in a survey conducted during the period of March-May 2019 in nurseries, warehouses, and shops in different locations(Alexandria, El-Behera, and Giza), in Egypt. The percentage of disease infection was the highest in Alexandria (47.67%) followed by Giza and El-Behera Governorates with 33.14% and 26.98%, respectively. In addition, the disease severity for the three surveyed governorates ranged between 35.19% (El-Behera Governorate) and 31.53% (Giza Governorate). These findings are not unexpected as disease infection and severity were of the highest values in such governorates with high humidity, Alexandria, and El-Behera Governorates. These results are in harmony with other investigators 2,3,8 .

Discussion
In the present work, five fungal species were found to be associated with the surveyed lucky bamboo samples showing root rot, basal stem rot, and wilt disease complex. These fungal species were R. solani, Fusarium oxysporum, F. solani, Aspergillus niger, and Alternaria alternata. Most of these fungi were isolated from lucky bamboo by several researchers 3-6 . However, R. solani isolates were the most dominant among fungal species recovered with a percentage of 80.89%. These findings also are in harmony with other investigators 10,11 . R. solani is one of the most dangerous and highly destructive fungal pathogens affecting many ornamental crops and several treatments and trials using natural products and bioagents have been done to control its growth 7,8,[59][60][61][62][63][64] .
Adoption of biological control is one of the crucial approaches currently at the forefront and is strongly desired for sustainable agriculture 18 . Several features of using biocontrol agents have been reported as one eco-friendly alternative or a supplemental way of reducing the use of toxic fungicides in ornamental plants 65 . Therefore, it is important to come up with new biological control products for eco-friendly and sustainable efficient benefits 17 . Furthermore, increasing levels of plant resistance using biological inducers isolated from the same plant is a new sustainable strategy for plant disease control. Identification of biocompatible isolates for managing lucky bamboo root rot, basal stem rot, and wilt caused by R. solani would be a beneficial contribution to disease management with low toxicity and minimal potential risk to the environment.
The present study supported this phenomenon, where four bioagents Clonostachys rosea, Bacillus circulans, B. siamensis, and Ochrobactrum anthropi were isolated from healthy lucky bamboo plants and were identified at the molecular level and significantly showed potential to inhibit R. solani in vitro as well as in vivo on lucky bamboo plants in vase treatments compared with the untreated inoculated control (negative control) as well as certain fungicides and biocides. All the tested bioagents (C. rosea, B. circulans, B. siamensis, and O. anthropi) as well as the tested fungicides and biocides, i.e., Moncut (2 g/l), Rizolex-T (2.0 g/l), Topsin-M (1 g/l), Bio-Zeid (2.5 g/l), and Bio-Arc (2.5 g/l), significantly inhibited the in vitro growth (colony diameter) of the tested R. solani isolate to different degrees.
Meanwhile, the in vivo experiment supported the in vitro results for the most effective treatments. All the tested treatments significantly decreased the percentage of infection and disease severity, showed significantly decreased disease incidence, and gave positive increasing responses regarding the treatment efficiency compared to the inoculated untreated control. The bioagent O. anthropi showed the highest effect, i.e., the lowest disease incidence and disease severity compared to the untreated inoculated control. This was not significantly different from the fungicide Moncut, and the bioagent C. rosea. These results are consistence with other investigators 14,15,28,[66][67][68][69] .
The bioagent fungus Clonostachys rosea (Gliocladium roseum) is proven to be a promising and strong biocontrol agent for a variety of plant pathogens including fungal, nematodes, and insects. It has been recorded as an aggressive parasite against many fungi of excellent bioagent in plant diseases through techniques like nutrient competition and hyperparasitism 47,70,71 . Also, mechanisms of C. rosea biocontrol are represented in the production of a wide range of volatile organic compounds which are toxic to organism's pathogens, and it could  O. anthropi, a siderophore-producing bacteria, is used as a potential biological control agent. It has shown a pretty antagonistic activity against many fungi such Botrytis cinerea, Colletotrichum orbiculare, and Fusarium oxysporum, etc. 66,68,74 .
Meanwhile, it has been indicated that Bacillus circulans emergence of chitinase enzyme activity against various plant pathogenic fungi, and also the B. spp. as a biocontrol has many benefits, including viz increasing mineral uptake, nitrogen fixation, and growing a strong and disease-resistant plant 28,41,75,76 . Additionally, B. siamensis succeeded as a biocontrol agent by producing antifungal compounds 29 such as poly-γ-glutamic acid 67 , and it was proven to enhance plant growth and heighten plant growth-promoting qualities 27 .
The most important findings of this research are the isolation and identification of the pathogenic fungus R. solani from the symptoms of natural infection of bamboo plants. Furthermore, isolation and identification of four  The present study supported the identification of biocompatible isolates for managing lucky bamboo root rot, and basal stem rot caused by R. solani that would be a beneficial contribution to disease management with low toxicity and minimal potential risk to the environment.

Conclusions
This is the first report of the isolation and identification of the pathogenic fungus Rhizoctonia solani from the symptoms of natural infection of bamboo, and, four associated biocontrol agents of the healthy lucky bamboo. They were identified based on morphological and microscopic characteristics, molecular phylogenetic analysis and GenBank accession. Consequently, the bioagents Ochrobactrum anthropi MW441317 at 1 × 10 8 CFU/ml, or Clonostachys rosea AUMC15121 at 1 × 10 7 /ml proved to be efficient to control the growth of R. solani which causes www.nature.com/scientificreports/